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Nonnanat Yooyuen. Overproduction of proteins of unknown function of prophage loci of Candidatus Liberibacter asiaticus an underlying cause of Huanglongbing disease in citrus plants. Master's Degree(Chemistry). Kasetsart University. Office of the University Library. : Kasetsart University, 2020.
Overproduction of proteins of unknown function of prophage loci of Candidatus Liberibacter asiaticus an underlying cause of Huanglongbing disease in citrus plants
Name: Nonnanat Yooyuen
Classification :.LCCS: QD431
LCSH:
Proteins
LCSH:
Greening disease
LCSH:
Rhizobiaceae
Abstract:
Citrus greening disease or Huanglongbing (HLB) is currently the most severe disease of citrus plants worldwide. The citrus greening disease is challenging to study and manage because the causal agent in Asia is Candidatus Liberitacter asiaticus (CLas), a fastidious phloem-limited bacterium, which cannot be cultivated in vitro. In this work, we focused on the Type-D prophage region of CLas correlating with blotchy mottle symptom of the HLB disease. Since the genome sequence of CLas (Thailand strain) was not available, the open reading frames (ORFs) larger than 300 nucleotides in the Type-D prophage region were identified using Nucleotide BLAST based on the sequencing data of CLas (Florida strain). Eight ORFs (orf2, orf3, orf6, orf7, orf8, orf9, orf10, and orf11) were successfully amplified from the total DNA extracted from the leave midribs of HLB-infected Citrus aurantifolia Swingle collected from Trang province, Thailand. The PCR products were then cloned into the pGEM-T easy vector. After DNA sequence analysis, there were mutations in all of the ORFs of Thailand strain when compared with Florida strain. Particularly, the careful analysis revealed mutations in orf6, resulting in the absence of this ORF in Thailand strain. Therefore, seven ORFs were subcloned into the pET28b expression vector for protein production. First, the heterologous toxicity of these proteins was evaluated. Overexpression of orf2, orf8, and orf10 was toxic to Escherichia coli strain BL21(DE3) host cells, while orf7 and orf9 did not exhibit toxic phenotype. Subsequently, seven orfs were subcloned into the pCXSN-HA expression vector, and Arabidopsis seedlings were co-cultivated with Agrobacterium tumefaciens strain GV2260 harboring the corresponding plasmids. The length of Arabidopsis seedlings appeared to be significantly shorter than the control experiments, except for orf2 and orf3. Then, all seven orfs were subjected to protein overexpression. The expression temperature was optimized; however, none of the seven proteins could be expressed and purified to homogeneity. Finally, the previously studied ORF5 was overexpressed and purified to homogeneity using Ni-NTA affinity chromatography followed by size exclusion chromatography. The protein was subjected to crystallization trials, and the crystal growth is currently in progress.
Kasetsart University. Office of the University Library
Address:
Bangkok
Email:
tdckulib@ku.ac.th
Name: Pitak Chuawong
Role:
Thesis Advisor
Name: Pakorn Wattana-Amorn
Role:
Thesis CO-Advisor
Role:
Thesis CO-Advisor
Created:
2020
Modified:
2025-07-15
Issued:
2025-07-15
วิทยานิพนธ์/Thesis
application/pdf
URL: https://www.lib.ku.ac.th/KUthesis/2563/nonnanat-yoo-all.pdf
CallNumber:
QD431 .N66
eng
DegreeName: Master of Science
Level: Master's Degree
Descipline: Chemistry
Grantor: Kasetsart University
©copyrights Kasetsart University
RightsAccess:
ใช้เวลา
0.020301 วินาที
Nonnanat Yooyuen
| Title | Contributor | Type |
|---|---|---|
| Overproduction of proteins of unknown function of prophage loci of Candidatus Liberibacter asiaticus an underlying cause of Huanglongbing disease in citrus plants
มหาวิทยาลัยเกษตรศาสตร์ Nonnanat Yooyuen | Pitak Chuawong | วิทยานิพนธ์/Thesis |
Pitak Chuawong
Pakorn Wattana-Amorn
Supachai Vuttipongchaikij